ELECTROCHEMICAL PROBES FOR DETECTION OF MOLECULAR INTERACTIONS AND DRUG DISCOVERY

• US 20020012943A1
• Filed: 02/06/1998
• Published: 01/31/2002
• Est. Priority Date: 02/06/1997
• Status: Abandoned Application

• Current Assignee: Xanthon, Inc. (Nokia Corporation), University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: THORP, H. HOLDEN, FOWLKES, DANA M.
• Application Number:
  US09/019,679
• Publication Number:
  US 20020012943A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: G01N 2333/02 Hepadnaviridae, e.g. hepatitis B virus
• CPC Class Codes: G01N 2333/162 env, e.g. gp160, gp110/120, gp41, V3, peptid T, DC4-Binding site
• CPC Class Codes: G01N 2333/36 from Actinomyces; from Streptomyces (G)
• CPC Class Codes: G01N 2333/42 Lectins, e.g. concanavalin, phytohaemagglutinin
• CPC Class Codes: G01N 2333/4727 Calcium binding proteins, e.g. calmodulin
• CPC Class Codes: G01N 2333/503 basic FGF [bFGF]
• CPC Class Codes: G01N 2333/70503 Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
• CPC Class Codes: G01N 2333/70546 Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
• CPC Class Codes: G01N 2333/7056 Selectin superfamily, e.g. LAM-1, GlyCAM, ELAM-1, PADGEM
• CPC Class Codes: G01N 2333/71 for growth factors; for growth regulators
• CPC Class Codes: G01N 2333/715 for cytokines; for lymphokines; for interferons
• CPC Class Codes: G01N 2333/745 Assays involving non-enzymic blood coagulation factors
• CPC Class Codes: G01N 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 2333/8107 Endopeptidase (E.C. 3.4.21-99) inhibitors
• CPC Class Codes: G01N 2333/9726 Tissue plasminogen activator
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

This invention relates to methods and apparati for performing electrochemical analyses. The invention provides an electrochemical apparatus for performing potentio metric analyses for detecting specific binding between a first member of a biological binding pair immobilized on an electrode and a second member of a biological binding pair that is electrochemically labeled, in the presence of an electrochemical mediator. Methods for using the apparatus of the invention for performing binding and competition binding assays are provided. The invention also provides methods for performing high throughput screening assays for detecting inhibition of specific binding between the members of the biological binding pair for use in drug development, biochemical analysis and protein purification assays.

Claims

1. An apparatus for performing an electrochemical assay for detecting binding between members of a biological binding pair, the apparatus comprising

a first electrode, wherein the electrode comprises a conducting or semiconducting material, and wherein the electrode has a surface that is coated with a porous, hydrophilic, polymeric layer to which a first member of the biological binding pair is immobilized thereto,

a second, reference electrode comprising a conducting metal in contact with an aqueous electrolyte solution,

a third, auxiliary electrode comprising a conducting metal

wherein each of the electrodes is electrically connected to a potentiostat, and wherein the apparatus further comprises

a reaction chamber containing a solution of an electrolyte, wherein each of the electrodes is in electrochemical contact therewith, the solution further containing

an electrochemical mediator comprising a chemical species capable of participating in a reduction/oxidation reaction with the electrodes under conditions whereby an electrical potential is applied to the electrodes, and wherein the solution further contains

a second member of the biological binding pair, wherein said second member is electrochemically labeled with a chemical species capable of participating in a reduction/oxidation reaction with the electrochemical mediator under conditions whereby an electrical potential is applied to the electrodes

wherein a current is produced in the apparatus when an electrical potential is applied to the electrodes under conditions wherein the second member of the biological binding pair is bound to the first member of the biological binding pair.

2. An apparatus according to claim 1, wherein the electrochemical assay is cyclic voltammetry.

3. An apparatus according to claim 1, wherein the apparatus further comprises a multiplicity of each of the electrodes and a multiplicity of reaction chambers, wherein each reaction chamber contains an electrolyte and is in electrochemical contact with one each of the three electrodes among the multiplicity of electrodes in the apparatus.

4. An apparatus according to claim 1, wherein the second member of the biological binding pair is electrochemically labeled with ruthenium.

5. An apparatus according to claim 1, wherein the electrochemical mediator is a ruthenium compound.

6. An apparatus according to claim 1, wherein the first member of the biological binding pair is a receptor protein or ligand binding fragment thereof and the second member of the biological binding pair is a ligand that specifically binds to the receptor protein.

7. An apparatus according to claim 1, wherein the first member of the biological binding pair is an antibody protein or antigen binding fragment thereof and the second member of the biological binding pair is an antigen that specifically binds to the antibody.

8. An apparatus according to claim 1, wherein the first member of the biological binding pair is a first protein or fragment thereof and the second member of the biological binding pair is a second protein or fragment thereof that specifically binds to the first protein.

15. An electrochemical mediator according to claim 1 comprising a ruthenium compound.

16. A method for detecting binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair immobilized on an electrode using an apparatus according to claim 1, the method comprising the steps of:

a) providing a first reaction chamber in electrochemical contact with each of the electrodes according to claim 1, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, and a second reaction chamber in electrochemical contact with each of the electrodes according to claim 1, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, each of the electrodes being electrically connected to a potentiostat;

wherein the first reaction chamber contains an electrochemical mediator according to claim 1 and an electrochemically-labeled second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, and wherein the second reaction chamber contains an electrochemical mediator according to claim 1 and an electrochemically-labeled species that does not specifically bind to the immobilized first member of the biological binding pair;

the method further comprising the steps of;

b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 1 to produce a current in the electrodes of the apparatus; and

c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein binding of the electrochemically labeled second member of the biological binding pair with the immobilized first member of the biological binding pair is detected by the production of a larger current in the first reaction chamber than is produced in the second reaction chamber.

17. A method for identifying an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair immobilized on an electrode using an apparatus according to claim 1, the method comprising the steps of:

a) providing a first reaction chamber in electrochemical contact with each of the electrodes according to claim 1, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, and a second reaction chamber in electrochemical contact with each of the electrodes according to claim 1, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, each of the electrodes being electrically connected to a potentiostat;

wherein each of the reaction chambers contains an electrochemical mediator according to claim 1 and an electrochemically-labeled second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, and wherein the second reaction chamber further contains an inhibitor of binding of the second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair;

the method further comprising the steps of;

b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 1 to produce a current in the electrodes of the apparatus; and

c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein an inhibitor of binding of the electrochemically labeled second member of the biological binding pair with the immobilized first member of the biological binding pair is identified by the production of a larger current in the first reaction chamber than is produced in the second reaction chamber.

18. A method for screening a complex chemical mixture for an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair immobilized on an electrode using an apparatus according to claim 1, the method comprising the steps of:

a) providing a first reaction chamber in electrochemical contact with each of the electrodes according to claim 1, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, and a second reaction chamber in electrochemical contact with each of the electrodes according to claim 1, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, each of the electrodes being electrically connected to a potentiostat;

wherein each of the reaction chambers contains an electrochemical mediator according to claim 1 and an electrochemically-labeled second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, and wherein the second reaction chamber further contains a portion of the complex mixture comprising an inhibitor of binding of the second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair;

the method further comprising the steps of;

b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 1 to produce a current in the electrodes of the apparatus; and

c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein a complex mixture having an inhibitor of binding of the electrochemically labeled second member of the biological binding pair with the immobilized first member of the biological binding pair is identified by the production of a larger current in the first reaction chamber than is produced in the second reaction chamber.

19. A method according to claim 16 wherein the second member of the biological binding pair is an electrochemically labeled surrogate ligand.

20. A method according to claim 17 wherein the second member of the biological binding pair is an electrochemically labeled surrogate ligand.

21. A method according to claim 18 wherein the second member of the biological binding pair is an electrochemically labeled surrogate ligand.

22. A method according to 18 comprising the additional steps of:

d) chemically fractionating the complex mixture having an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair immobilized on the first electrode, to produce fractionated submixtures; and

e) performing steps (a) through (c) of the method of claim 18 on each of the fractionated submixtures to identify the submixtures that have an inhibitor of binding of the biological binding pair.

9. An electrode comprising a conducting or semiconducting material, wherein the electrode has a surface that is coated with a porous, hydrophilic, polymeric layer to which a first member of a biological binding pair is immobilized thereto.

10. A kit for preparing an electrode according to claim 9, wherein the kit comprises an electrode comprising a conducting or semi-conducting material, a first member of a biological binding pair, a reagent for preparing a porous, hydrophilic, polymeric layer on the surface of the electrode, and a reagent for immobilizing the first member of the biological binding pair within the porous, hydrophilic, polymeric layer on the surface of the electrode.

11. A method for preparing an electrode according to claim 9, comprising the steps of:

a) providing an electrode comprising a conducting or semi-conducting material;

b) preparing a porous, hydrophilic, polymeric layer on the surface of the electrode; and

c) immobilizing a first member of a biological binding pair within the porous, hydrophilic, polymeric layer on the surface of the electrode.

12. An electrochemically labeled surrogate ligand comprising a second member of a biological binding pair having a binding affinity of from about 1 nanomolar (nM) to about 100 micromolar (μ

M) for a first member of a biological binding pair.

13. An electrochemically labeled surrogate ligand according to claim 12, labeled with a ruthenium compound.

14. An electrochemically labeled surrogate ligand according to claim 12, comprising a peptide of formula:

Gly-His-Gly-Ser-Gly-Arg-Ala-Leu-Pro-Pro-Leu-Pro-Arg-Tyr-NH₂ 

 

(SEQ ID No.;

     1);

His-Gly-Ser-Gly-Ser-Phe-Ser-His-Pro-Gln-Asn-Thr 

 

(SEQ ID No.;

     2);

biotin-His-His-Ser-Gly-Ser-Gly-Ser-Gln-Thr-Phe-Ser-Asp-Leu-Trp-Lys-Leu 

 

(SEQ ID No.;

     3),Arg-Pro-Leu-Pro-Pro-Leu-Pro 

 

(SEQ ID No.;

     4),Ala-Pro-Pro-Val-Pro-Pro-Arg 

 

(SEQ ID No.;

     5),

or

Leu-Tyr-Ser-Trp-Pro-Asp-Glu-Gln-Tyr-Glu-Arg-Pro-Ser-Gly-Ser-Gly-Lys 

 

(SEQ ID No.;

     6)

23. An apparatus for performing an electrochemical assay for detecting binding between members of a biological binding pair, the apparatus comprising

a first electrode, wherein the electrode comprises a conducting or semiconducting material, and wherein the electrode has a surface that is coated with a porous, hydrophilic, polymeric layer, wherein a first member of the biological binding pair and an electrochemical mediator comprising a chemical species capable of participating in a reduction/oxidation reaction with the electrodes under conditions whereby an electrical potential is applied to the electrodes, are each immobilized thereto,

a second, reference electrode comprising a conducting metal in contact with an aqueous electrolyte solution,

a third, auxiliary electrode comprising a conducting metal

wherein each of the electrodes is electrically connected to a potentiostat, and wherein the apparatus further contains

a reaction chamber containing a solution of an electrolyte, wherein each of the electrodes is in electrochemical contact therewith, the solution further comprising

a second member of the biological binding pair, wherein said second member is electrochemically labeled with a chemical species capable of participating in a reduction/oxidation reaction with the electrochemical mediator under conditions whereby an electrical potential is applied to the electrodes

wherein a current is produced in the apparatus when an electrical potential is applied to the electrodes under conditions wherein the second member of the biological binding pair is bound to the first member of the biological binding pair.

24. An apparatus according to claim 23, wherein the electrochemical assay is cyclic voltammetry.

25. An apparatus according to claim 23, wherein the apparatus further comprises a multiplicity of each of the electrodes and a multiplicity of reaction chambers, wherein each reaction chamber contains an electrolyte and is in electrochemical contact with one each of the three electrodes among the multiplicity of electrodes in the apparatus.

26. An apparatus according to claim 23, wherein the second member of the biological binding pair is electrochemically labeled with ruthenium.

27. An apparatus according to claim 23, wherein the electrochemical mediator is an osmium compound.

28. An apparatus according to claim 23, wherein the electrochemical mediator is a ruthenium compound.

29. An apparatus according to claim 23, wherein the first member of the biological binding pair is a receptor protein or ligand binding fragment thereof and the second member of the biological binding pair is a ligand that specifically binds to the receptor protein.

30. An apparatus according to claim 23, wherein the first member of the biological binding pair is an antibody protein or antigen binding fragment thereof and the second member of the biological binding pair is an antigen that specifically binds to the antibody.

31. An apparatus according to claim 23, wherein the first member of the biological binding pair is a first protein or fragment thereof and the second member of the biological binding pair is a second protein or fragment thereof that specifically binds to the first protein.

35. A method for detecting binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair immobilized on an electrode using an apparatus according to claim 23, the method comprising the steps of:

a) providing a first reaction chamber in electrochemical contact with each of the electrodes according to claim 23, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, and a second reaction chamber in electrochemical contact with each of the electrodes according to claim 23, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, each of the electrodes being electrically connected to a potentiostat;

wherein the first reaction chamber contains an electrochemically-labeled second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, and wherein the second reaction chamber contains an electrochemically-labeled species that does not specifically bind to the immobilized first member of the biological binding pair;

the method further comprising the steps of;

b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 23 to produce a current in the electrodes of the apparatus; and

c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein binding of the electrochemically labeled second member of the biological binding pair with the immobilized first member of the biological binding pair is detected by the production of a larger current in the first reaction chamber than is produced in the second reaction chamber.

36. A method for identifying an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair immobilized on an electrode using an apparatus according to claim 23, the method comprising the steps of:

a) providing a first reaction chamber in electrochemical contact with each of the electrodes according to claim 23, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, and a second reaction chamber in electrochemical contact with each of the electrodes according to claim 23, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, each of the electrodes being electrically connected to a potentiostat;

wherein each of the reaction chambers contains an electrochemically-labeled second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, and wherein the second reaction chamber further contains an inhibitor of binding of the second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair;

the method further comprising the steps of;

b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 23 to produce a current in the electrodes of the apparatus; and

c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein an inhibitor of binding of the electrochemically labeled second member of the biological binding pair with the immobilized first member of the biological binding pair is identified by the production of a larger current in the first reaction chamber than is produced in the second reaction chamber.

37. A method for screening a complex chemical mixture for an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair immobilized on an electrode using an apparatus according to claim 23, the method comprising the steps of:

a) providing a first reaction chamber in electrochemical contact with each of the electrodes according to claim 23, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, and a second reaction chamber in electrochemical contact with each of the electrodes according to claim 23, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, each of the electrodes being electrically connected to a potentiostat;

wherein each of the reaction chambers contains an electrochemically-labeled second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, and wherein the second reaction chamber further contains a portion of the complex mixture comprising an inhibitor of binding of the second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair;

the method further comprising the steps of;

b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 23 to produce a current in the electrodes of the apparatus; and

c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein a complex mixture having an inhibitor of binding of the electrochemically labeled second member of the biological binding pair with the immobilized first member of the biological binding pair is identified by the production of a larger current in the first reaction chamber than is produced in the second reaction chamber.

38. A method according to claim 35 wherein the second member of the biological binding pair is an electrochemically labeled surrogate ligand.

39. A method according to claim 36 wherein the second member of the biological binding pair is an electrochemically labeled surrogate ligand.

40. A method according to claim 37 wherein the second member of the biological binding pair is an electrochemically labeled surrogate ligand.

41. A method according to claim 37 comprising the additional steps of:

d) chemically fractionating the complex mixture having an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair immobilized on the first electrode, to produce fractionated submixtures; and

e) performing steps (a) through (c) of the method of claim 37 on each of the fractionated submixtures to identify the submixtures that have an inhibitor of binding of the biological binding pair.

32. An electrode comprising a conducting or semiconducting material, wherein the electrode has a surface that is coated with a porous, hydrophilic, polymeric layer and wherein a first member of the biological binding pair and an electrochemical mediator comprising a chemical species capable of participating in a reduction/oxidation reaction with the electrode under conditions whereby an electrical potential is applied to the electrode, are each immobilized thereto.

33. A kit for preparing an electrode according to claim 32, wherein the kit comprises an electrode comprising a conducting or semi-conducting material, an electrochemical mediator, a first member of a biological binding pair, a reagent for preparing a porous, hydrophilic, polymeric layer on the surface of the electrode, and a reagent for immobilizing the first member of the biological binding pair and the electrochemical mediator within the porous, hydrophilic, polymeric layer on the surface of the electrode.

34. A method for preparing an electrode according to claim 32, comprising the steps of:

a) providing an electrode comprising a conducting or semi-conducting material;

b) preparing a porous, hydrophilic, polymeric layer on the surface of the electrode; and

c) immobilizing a first member of a biological binding pair and the electrochemical mediator within the porous, hydrophilic, polymeric layer on the surface of the electrode.

42. An apparatus for performing an electrochemical assay for detecting binding between members of a biological binding pair, the apparatus comprising

a first electrode, wherein the electrode comprises a conducting or semiconducting material, and wherein the electrode has a surface that is coated with a porous, hydrophilic, polymeric layer, wherein a first member of the biological binding pair and an electrochemical mediator comprising a chemical species capable of participating in a reduction/oxidation reaction with the electrodes under conditions whereby an electrical potential is applied to the electrodes, are each immobilized thereto,

a second, reference electrode comprising a conducting metal in contact with an aqueous electrolyte solution,

a third, auxiliary electrode comprising a conducting metal

wherein each of the electrodes is electrically connected to a potentiostat, and wherein the apparatus further comprises

a reaction chamber containing a solution of an electrolyte, wherein each of the electrodes is in electrochemical contact therewith, the solution further comprising

a second member of the biological binding pair, wherein said second member is bound to an electrochemical catalyst capable of participating in a reduction/oxidation reaction with the electrochemical mediator under conditions whereby an electrical potential is applied to the electrodes, wherein the electrolyte in the reaction chamber further contains a substrate for the electrochemical catalyst

wherein a current is produced in the apparatus when an electrical potential is applied to the electrodes under conditions wherein the second member of the biological binding pair is bound to the first member of the biological binding pair in the presence of the substrate for the electrochemical catalyst bound to the second member of the biological binding pair.

43. An apparatus according to claim 42, wherein the electrochemical assay is chronoamperometry.

44. An apparatus according to claim 42, wherein the apparatus further comprises a multiplicity of each of the electrodes and a multiplicity of reaction chambers, wherein each reaction chamber contains an electrolyte and is in electrochemical contact with one each of the three electrodes among the multiplicity of electrodes in the apparatus.

45. An apparatus according to claim 42, wherein the electrochemical catalyst bound to the second member of the biological binding pair is an enzyme.

46. An apparatus according to claim 45, wherein the enzyme is horse radish peroxidase.

47. An apparatus according to claim 42, wherein the electrochemical mediator is an osmium compound.

48. An apparatus according to claim 42, wherein the first member of the biological binding pair is a receptor protein or ligand binding fragment thereof and the second member of the biological binding pair is a ligand that specifically binds to the receptor protein.

49. An apparatus according to claim 42, wherein the first member of the biological binding pair is an antibody protein or antigen binding fragment thereof and the second member of the biological binding pair is an antigen that specifically binds to the antibody.

50. An apparatus according to claim 42, wherein the first member of the biological binding pair is a first protein or fragment thereof and the second member of the biological binding pair is a second protein or fragment thereof that specifically binds to the first protein.

51. A method for detecting binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair immobilized on an electrode using an apparatus according to claim 42, the method comprising the steps of:

a) providing a first reaction chamber in electrochemical contact with each of the electrodes according to claim 42, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, and a second reaction chamber in electrochemical contact with each of the electrodes according to claim 42, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, each of the electrodes being electrically connected to a potentiostat;

wherein the first reaction chamber contains a substrate for the electrochemical catalyst and a second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, bound to an electrochemical catalyst, and wherein the second reaction chamber contains a substrate for the electrochemical catalyst and a chemical species that does not specifically bind to the immobilized first member of the biological binding pair, bound to an electrochemical catalyst;

the method further comprising the steps of;

b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 42 to produce a current in the electrodes of the apparatus; and

c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein binding of the second member of the biological binding pair with the immobilized first member of the biological binding pair is detected by the production of a larger current in the first reaction chamber than is produced in the second reaction chamber.

52. A method for identifying an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair immobilized on an electrode using an apparatus according to claim 42, the method comprising the steps of:

a) providing a first reaction chamber in electrochemical contact with each of the electrodes according to claim 42, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, and a second reaction chamber in electrochemical contact with each of the electrodes according to claim 42, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, each of the electrodes being electrically connected to a potentiostat;

wherein each of the reaction chambers contains a substrate for the electrochemical catalyst and a second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, bound to an electrochemical catalyst, and wherein the second reaction chamber further contains an inhibitor of binding of the second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, and further comprising a substrate for the electrochemical catalyst;

the method further comprising the steps of;

b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 42 to produce a current in the electrodes of the apparatus; and

c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein an inhibitor of binding of the second member of the biological binding pair with the immobilized first member of the biological binding pair is identified by the production of a larger current in the first reaction chamber than is produced in the second reaction chamber.

53. A method for screening a complex chemical mixture for an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair immobilized on an electrode using an apparatus according to claim 42, the method comprising the steps of:

a) providing a first reaction chamber in electrochemical contact with each of the electrodes according to claim 42, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, and a second reaction chamber in electrochemical contact with each of the electrodes according to claim 42, wherein the first electrode comprises a first member of the biological binding pair immobilized thereto, each of the electrodes being electrically connected to a potentiostat;

wherein each of the reaction chambers contains a substrate for the electrochemical catalyst and a second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair, bound to an electrochemical catalyst, and wherein the second reaction chamber further contains a portion of the complex mixture comprising an inhibitor of binding of the second member of the biological binding pair that specifically binds to the immobilized first member of the biological binding pair and further comprising a substrate for the electrochemical catalyst;

the method further comprising the steps of;

b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 42 to produce a current in the electrodes of the apparatus; and

c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein a complex mixture having an inhibitor of binding of the second member of the biological binding pair with the immobilized first member of the biological binding pair is identified by the production of a larger current in the first reaction chamber than is produced in the second reaction chamber.

54. A method according to claim 51 wherein the second member of the biological binding pair is a surrogate ligand.

55. A method according to claim 52 wherein the second member of the biological binding pair is a surrogate ligand.

56. A method according to claim 53 wherein the second member of the biological binding pair is a surrogate ligand.

57. A method according to claim 53 comprising the additional steps of:

d) chemically fractionating the complex mixture having an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair immobilized on the first electrode, to produce fractionated submixtures; and

e) performing steps (a) through (c) of the method of claim 53 on each of the fractionated submixtures to identify the submixtures that have an inhibitor of binding of the biological binding pair.

58. A method according to claim 55 wherein the inhibitor is added to the second reaction chamber after the surrogate ligand is added to the second reaction chamber.

59. A method according to claim 55 wherein the inhibitor is added to the second reaction chamber before the surrogate ligand is added to the second reaction chamber.

60. A method according to claim 55 wherein the inhibitor is added to the second reaction chamber together with the surrogate ligand is added to the second reaction chamber.